Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects over 350 million people worldwide and causes 600,000 deaths annually. Current therapies for HBV infection use interferon 1 or one of five nucleoside analogs. Neither form of treatment cures HBV infections and both have major limitations. Furthermore, the nucleoside analogs all target the DNA polymerase active site on the viral reverse transcriptase, and hence cross-resistance between some of the drugs is common. Therefore, novel antivirals that act on targets other than the DNA polymerase active site are urgently needed. We recently identified two motifs called "T3" and "RT1" on the Duck Hepatitis B virus (DHBV) reverse transcriptase that together form the site where the enzyme first binds to the RNA form of the viral genome. Disrupting RNA binding at the T3:RT1 site blocks reverse transcription. Neither T3 nor RT1 are in the DNA polymerase active site, and consequently inhibitors of the T3:RT1 site would function by a novel mechanism and hence would be perfect candidates for combination therapy with the nucleoside analog drugs. The T3 and RT1 motifs were identified through their high homology between DHBV and HBV, and mutating the HBV T3 motif ablates DNA synthesis, but little work has been done with the HBV enzyme itself. Therefore, in this Phase I STTR project we will extend our analysis of RNA binding by the DHBV reverse transcriptase to the HBV enzyme. The goal of this application is to identify the form of the HBV enzyme best suited for subsequent high through-put screening for novel anti-T3:RT1 drugs. Aim 1. Evaluate the role of the HBV T3 and RT1 motifs in RNA binding. In vitro RNA binding assays with recombinant HBV reverse transcriptase will be done to determine how well data obtained with the DHBV enzyme reflects RNA binding at the HBV T3:RT1 site. These results will be confirmed in vivo by evaluating the effects of mutations to the HBV T3 and RT1 motifs on encapsulation of the HBV genomic RNA. Aim 2. Determine the optimal configuration for an HBV reverse transcriptase RNA binding assay amenable to drug screening. We will adapt our current filter-binding assay that measures RNA binding by the DHBV reverse transcriptase to the medically-relevant HBV enzyme. In Phase II of this STTR project we will fully develop an HBV reverse transcriptase RNA binding assay into a 384-well format employing fluorescent detection of the RNA and conduct a pilot screen of negatively-charged non-nucleoside compounds for inhibitors of the RNA binding reaction. We will then seek a major pharmaceutical partner for full-scale high-throughput screening. PUBLIC HEALTH RELEVANCE: Hepatitis B virus replicates by reverse transcription, kills 600,000 people each year, and current therapies for the infection are inadequate. Using an animal model for HBV, we recently identified the site on the viral reverse transcriptase where the enzyme first binds to its viral RNA template and demonstrated that disrupting this site blocks viral replication. Here, we will extend these results to the medically-relevant HBV enzyme, with the goal of identifying the format of an assay best suited for subsequent high through-put screening for novel anti-HBV drugs.